While exerting their motile function, molecular motors experience various extents of mechanical load that regulates their mechanical output and also plays a role in the coordination between units of supramolecular assemblies. In this study, we were able to investigate the load dependence of the kinetics of non-muscle myosin II isoforms in solution conditions utilizing the intramolecular strain that arises when myosin binds via both heads to adjacent sites on the actin filament. We confirm this binding arrangement for acto-non-muscle myosin II by negative staining electron microscopy. Using transient kinetic methods, we show that nucleotide binding is not strain-sensitive in these myosins, but ADP release exhibits a marked strain effect, which will, in turn, regulate the overall mechanochemical properties. This feature provides a basis for cellular functions such as a mechanical response to load and/or changes in intracellular ADP concentration, and a possible load-dependent stalling of these cytoplasmic motors.